5% formaldehyde solution and stained with ammoniacal silver nitrate solution [0.15% (w/v) silver nitrate, 0.05% (w/v) sodium hydroxide and 2.5% (v/v) ammonium hydroxide]. SDS-PAGE analysis CX-5461 ic50 comparing egg proteins from E. tuberculatum queens and workers showed differences
between the castes. The eggs of the workers contained two major proteins with molecular weights of 31 and 156 kDa, while the eggs of queens had eight major proteins, four of which (31, 36, 123, and 156 kDa) appeared with greater intensity than the others (81, 86, 96, and 101 kDa) ( Fig. 1). Haemolymph samples from workers of different ages displayed different protein patterns. Proteins with MWs of 43, 84, 89, and 195 kDa occurred in samples from workers of all ages (Fig. 1). The haemolymph of workers aged 2 and 5 days had a 120 kDa protein that was not found in workers of other ages. Haemolymph from workers with 10 days of age showed small quantities of the proteins of MWs 31 and 156 kDa present in worker oocytes (Fig. 1). These proteins also appeared in the haemolymph of workers aged 15,
20, 30, and 60 days. From the age of 20 days, all ants expressed the proteins of 38, 71, and 135 kDa. selleck products Workers 100 days of age did not show the proteins of 31 and 156 kDa (Fig. 1). In the haemolymph of queens, proteins of 85, 135, 156 and 195 kDa appeared in greater quantity, while the 31 and 43 kDa proteins were slightly detected (Fig. 1). To verify the presence of vitellogenin in ants of different ages, the two most abundant proteins in eggs of queens (Fig. 1) were isolated and used to immunize rabbits for antibody production. The antibodies obtained to proteins of 123 and 156 kDa were termed vg1 and vg2, respectively. Immunolocalization tests were performed to provide indirect evidence that the isolated proteins may correspond to vitellogenin. Immunohistochemistry (Fig. 2A–C) and immunofluorescence (Fig. 2D–F) showed positive reactions for antibodies vg1 and vg2 in fat body cells and oocytes. The fat body was characterized by large cells
that had a central nucleus Immune system and many vacuoles (Fig. 2A–C). The immunostained granules were found around these vacuoles and were clustered at the cell periphery (Fig. 2A and B). In the oocytes, the positive granules were observed throughout the ooplasm (Fig. 2E). The antibodies vg1 and vg2 were used in Western blot analysis of egg extracts from queens and workers, while the haemolymph samples were analyzed using only the vg2 antibody. The analysis showed that both antibodies reacted positively to the proteins of 123 and 156 kDa and also for smaller unspecific fragmentation products (Fig. 3). Analysis of the haemolymph showed a positive reaction to a protein of 156 kDa in samples from queens and workers aged 5, 10, 15, 20, 30, and 60 days (Fig. 4). An increase in the intensity of the reaction was obtained in samples from workers aged 20 and 30 days.